Methods and compositions to generate unique sequence dna probes, labeling of dna probes and the use of these probes

ABSTRACT

The invention relates generally to the field of the identification of DNA sequences, genes or chromosomes. Methods and composition to obtain Unique Sequence DNA probes are provided. Compositions comprised of and double stranded DNA containing Unique Sequences from which the repetitive sequences are eliminated according to the method described in this invention. The invention also relates to the preservation of cells that have been identified after immunomagnetic selection and fluorescent labeling in order to further interrogate the cells of interest. Furthermore the invention relates to genetic analysis of cells that have been identified after immunomagnetic selection and fluorescent labeling.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/067,532 filed on Oct. 10, 2008, which is a national stageapplication of PCT/US06/36656 filed on Sep. 20, 2006, which claimspriority to U.S. Provisional Applications 60/713,676, filed Sep. 20,2005; 60/729,536, filed Oct. 24, 2005; and 60/786,117, filed Mar. 27,2006, all of which are hereby incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates generally to the field of identification of DNAsequences, genes or chromosomes.

Generation of DNA Probes

Human genomic DNA is a mixture of unique sequences and repetitivesequences that are present in multiple copies throughout the genome. Insome applications, nucleic acid hybridization probes to detectrepetitive sequences are desirable. These probes have shown utility inthe fields of fetal cell diagnostics, oncology, and cytogenetics. Inother applications it is desirable to generate hybridization probes thatanneal only to unique sequences of interest on a chromosome. Preparationof unique sequence probes is confounded by the presence of numerousclasses of repetitive sequences throughout the genome of the organism(Hood et al., Molecular Biology of Eucaryotic Cells (Benjamin/CummingsPublishing Company, Menlo Park, Calif. 1975). The presence of repetitivesequences in hybridization probes will reduce the specificity of theprobes because portions of the probe will bind to other repetitivesequences found outside the sequence of interest. Thus, to ensurebinding of hybridization probes to a specific sequence of interest,efforts must be made to ensure that repetitive sequences in the probe donot anneal to the target DNA outside the sequence of interest.

Recent contributions have addressed this question by inhibitinghybridization of the repetitive sequences with the use of unlabeledblocking nucleic acids (U.S. Pat. No. 5,447,841 and U.S. Pat. No.6,596,479). Use of blocking nucleic acids in hybridizations isexpensive, does not completely prevent hybridization of the repetitivesequences, and can distort genomic hybridization patterns (Newkirk etal., “Distortion of quantitative genomic and expression hybridization byCot-1 DNA: mitigation of this effect,” Nucleic Acids Res. vol 33(22):e191 (2005)). Thus, methods that prevent hybridization of repeatsequences without the use of blocking DNA are necessary for optimalhybridization.

One means to achieve this is to remove unwanted repeat segments from thehybridization probes prior to hybridization. Techniques involving theremoval of highly repetitive sequences have been previously described.Absorbents, like hydroxyapatite, provide a means to remove highlyrepetitive sequences from extracted DNA. Hyroxyapatite chromatographyfractionates DNA on the basis of duplex re-association conditions, suchas temperature, salt concentration, or other stringencies. Thisprocedure is cumbersome and varies with different sequences. Repeat DNAcan also be removed by hybridization to immobilized DNA (Brison et al.,“General Methods for Cloning Amplified DNA by Differential Screeningwith Genomic Probes,” Molecular and Cellular Biology, Vol. 2, pp.578-587 (1982)). In all of these procedures, the physical removal of therepetitive sequences will depend upon the strict optimization ofconditions with inherent variations based upon the base composition ofthe DNA sequence.

Several other methods to remove repetitive sequences from hybridizationprobes have been described. One method involves using a cross-linkingagent to cross-link repetitive sequences either to directly preventhybridization of repetitive sequences or to prevent amplification ofrepeat sequences in a PCR reaction. (U.S. Pat. No. 6,406,850). Anothermethod uses PCR assisted affinity chromatography to remove repeats fromhybridization probes (U.S. Pat. No. 6,569,621). Both of these methodsrely on the use of labeled DNA to remove repeat sequences which makesthese processes complex and difficult to reproduce. Further, bothmethods are time consuming, requiring multiple rounds of repeat removalto produce functional probes, suitable for use in fluorescent in situhybridization (FISH) or other hybridization reactions requiring hightarget specificity.

The use of duplex specific nucleases which preferentially cleave doublestranded deoxyribonucleic acid molecules has been described for sequencevariant detection applications such as single nucleotide polymorphisms(US 2005/0164216; U.S. Pat. No. 6,541,204). The ability of the enzyme topreferentially cleave perfectly matched nucleic acid duplexpolynucleotides as compared to single stranded provides a means forremoving non-target double stranded DNA from the sample mixture.

The ability of these nucleases to specifically digest the duplex form ofpolynucleotides was discovered in the instant invention to providesubstantial benefit in manufacturing unique target specific probes thatdo not require blocking DNA, thus eliminating the costs and interferingaffect of blocking DNA, and providing a means for rapid, efficient andcost effective production of high specificity probes.

Detection of specific sequences in a genome makes use of the fact thatDNA consists of a helix of two DNA strands and that this double strandis most stable when these two strands are homologues. The DNA consistsof a phosphate-sugar phosphate backbone and to every sugar one of fourdifferent nitrogenous bases, cytosine guanine thymine or adenine, mightbe present. Homologue strands pair every cytosine with a guanine andevery thymine with an adenine. When a labeled homologue sequence isadded to a genome and the DNA is made single stranded, these labeledsequences will hybridize, under the right circumstances, to the specifichomologue sequence in the genome. For this in situ hybridization, anumber of probes are available for different detections purposes andapplications.

Whole Chromosome/Paint Probes (WCP)

WCP incorporates labeled DNA material, homologous to a specificchromosome. The material is obtained by flow sorting of metaphasechromosomes or by laser dissection from a metaphase spread which isamplified by PCR or a related technique. After labeling and applying itto a properly prepared nucleus, it will stain the target chromosome.However, such labeled probes will in addition stain other non-targetchromosomes because of structural or repetitive sequence elements thatare shared among some or all chromosomes.

Accordingly in order to stain only those sequences originating from theintended chromosome of interest, these common repetitive elements areusually inhibited by hybridization with blocking DNA or other methodsthat block or remove non-specific interactions.

Multiple chromosome paints are also applied to a single nucleus. WCP arelabeled by different fluorochromes or with a combination offluorochromes, providing no limit to the amount of WCP's applied in asingle hybridization. WCP's are mainly used for karyotyping and to studytranslocations of large fragments, regions and subregions of chromosomeswhich are best observed in a metaphase spread of a nucleus.

Centromere Probes

Centromere probes are targeted to a 171 bp sequence that occurs inrepetitive order in every centromeric region of the human chromosomes.All chromosomes have a slightly different sequence and because of thisall chromosomes are detected separately when the right hybridizationstringency is used. Only two chromosome pairs, 13 with 21 and 14 with22, share the same repeat and cannot be detected independently.Generally, centromeric probes are produced from plasmids containing aninsert from one or a few copies of the 171 pb repeat. These probes areable to be hybridized without the addition of blocking DNA because the171 bp sequences do not occur outside the regions of interest.

Telomere Probes

Human telomeres consist of an array of short repetitive sequences (i.e.TTAGGG). This is repeated several times in different amounts for everychromosome and individual test subject age. This repetitive sequence isused as a probe that will stain all chromosomes although not everychromosome will stain equally strong. To detect the telomeric end ofchromosomes, mostly a sub-telomeric bacterial artificial chromosome(BAC) clone is used. This BAC clone contains repetitive sequences whichshould be blocked or removed during or before hybridization.

Comparative Genomic Hybridization (CGH) Probes

CGH is a process that involves hybridizing a test genome to a referencegenome. The reference genome may take the form of a metaphase chromosomespread from a healthy individual or may be array based using probesequences that represent all or part of a genome. Microarray probes madeusing BAC clones contain repetitive sequences which must be blockedprior to hybridization. However, blocking has the potential to cause adeviation in the results when compared to repeat depleted probes (Knoland Rogan, Nucleic Acids Research, 2005, Vol. 33, No. 22). Further, theblocking step increases the cost of hybridization assays. If the probesequences are depleted of repetitive sequences, the blocking step of thelabeled genomic DNA is not necessary, resulting in a reduction in thecost and removal of any variation.

Gene Specific Probes

Gene specific probes are designed to detect a region of the genomecontaining a target gene or group of genes. These probes are used todetect amplifications or deletions of specific genomic areas whichcorrelated to the expression level of the specific gene of interest. Thecoding sequence of the gene(s) itself is not large enough to generate adetection signal for the probe that is visible using standardfluorescence microscope. Therefore such gene specific probes are notlimited to just the coding gene sequences (exons) but also involvenon-coding (introns), regulatory or other sequences around the gene.Because of the large sequences encompassed within even a gene specificprobe design they often suffer from the undesirable inclusion ofunwanted repetitive sequences. When such material is then either labeledand used in hybridizations or used in hybridizations and then labeled,the unwanted sequences must be blocked or removed from the probe to beable to detect the gene area specifically.

Microarray Probes

Similar to CGH, microarray probes are fixed to a carrier. In general,automated robotic techniques are used to spot cDNA-PCR products orsynthetic oligonucleotides on a slide or similar fixed surface. Also,techniques exist to synthesize sequences directly on a slide(Affimetrix, Inc, Santa Clara). The slides are hybridized with labeledcDNA or RNA in combination with different labeled cDNA or RNA ascontrols.

Coupling Reporter Molecules to DNA Probes

DNA probes are visualized by coupled reporter molecules. These moleculesneed to be incorporated in or attached to the DNA probe. One methodutilizes a reporter molecule, having nucleotides linked to enzymaticreactions. Examples include incorporation by nick translation or arandom prime reaction. Further, an amine coupled nucleotide, built inthis way, is subsequently coupled directly or indirectly to reportermolecules. Coupling is done by chemical labeling of the DNA. An exampleis the coupling of a reporter molecule linked to a platinum group whichforms a coordinative bond to the N7 position of guanine as used in ULSlabeling (Kreatech Diagnostics, Amsterdam) and described in U.S. Pat.No. 5,580,990; U.S. Pat. No. 5,714,327; U.S. Pat. No. 5,985,566; U.S.Pat. No. 6,133,038; U.S. Pat. No. 6,248,531; U.S. Pat. No. 6,338,943;U.S. Pat. No. 6,406,850; and U.S. Pat. No. 6,797,818. Reporter moleculescan be radioactive isotope, non-isotopic labels, digoxygenin, enzymes,biotin, avidin, streptavidin, luminescent agents such asradioluminescent, chemiluminescent, bioluminescent, andphotoluminescent, (including fluorescent and phosphorescent), dyes,haptens, and the like.

Sample Preparation

To be able to detect the labeled probes bound to interphase chromosomes,the nucleus should maintain morphology during and after the FISHprocedures. Using fixation, cells or nuclei are attached to a solidlayer such as a microscope slide. Fixation before during or afterattachment to the solid layer, provide reference for identification.Depending on the type of cell or tissue, the nuclei have to beaccessible for probe DNA, usually by pre-treating with proteolyticenzymes, heat, alcohols, denaturants, detergent solutions or acombination of treatments. Probe and nucleic DNA are made singlestranded by heat or alkali treatment and then allowed to hybridize.

Use of DNA Probes Microarrays

One common use of microarrays is to determine the RNA expression profileof a suspect tissue, tumor, or microbe. By analyzing the RNA expressionprofile, a prognosis for the treatment and survival of the patient isproposed. The prognostic value of RNA microarrays for clinical usage hasyet to be determined. Another common use of microarrays are array basedCGH. With this technique an entire genome can be screened foramplifications and/or deletions of chromosomal regions

Microscopy

Cytogenetic analysis in pre and post natal testing is used to determinewhether or not a fetus has a cytogenetic abnormality in a cellpopulation from the fetus. Samples are frequently obtained throughamniocenthesis, conducted in pregnant women who are considered to havean increased risk for cytogenetic abnormalities. Accordingly, thesecells are investigated for cytogenetic abnormalities. The same type ofinvestigations are performed to confirm cytogenetic abnormalities orinvestigate suspect cytogenetic abnormalities in cell populationsobtained after delivery.

Assessing Fetal Cells in Maternal Blood

During pregnancy, fetal cells may enter into the maternal blood withincreases in the number of these fetal cells found with trauma,(pre)-ecclampsy and abnormal pregnancies. In routine assessments offetomaternal hemorrhages, the frequently used Kleihauer-Betke test isbased on the detection of red blood cells expressing fetal hemoglobin.For detection of cytogenetic abnormalities, nucleated cells frommaternal blood are needed. The frequency of these cells is considerablylower and are estimated to be in the range of 1-10 fetal cells per mL ofmaternal blood. Nucleated red blood cells, trophoblast cells and thepresence of hematopoeitic progenitors that are of fetal origin provide atarget for isolation and probe hybridization in the detection ofcytogenetic abnormalities early in the pregnancies. To date a reliableand reproducible method to identify and assess the cytogeneticcomposition of these cells is not available. One of the main problemswith this analysis is the loss of fetal cells at various stepsthroughout the procedure, resulting in inconsistent or inconclusiveinformation.

Oncology

FISH is used to detect various kinds of chromosomal aberrations liketranslocations, deletions, amplifications, inversions, and duplications.These aberrations are detected in all types of cells and tissue. Inleukemia, cells are isolated from blood or bone marrow for subsequentFISH analysis. In bladder cancer, cells are isolated from urine. Cellsfrom solid tumors are obtained by puncture or excision of the tumoritself. Also, cells that are released by solid tumors are isolated fromthe blood and analyzed by FISH. The latter gives the opportunity tomonitor tumor treatment closely in order to detect a chromosomal changein the tumor. In some types of cancer, FISH provides a prognosis oftumor progression or predicts the efficacy of specific medication.Commercially, the most used FISH tests are the BCR-ABL translocationFISH in Chronic myclogenous leukemia and the her2/neu gene amplificationFISH in breast cancer.

Disseminated Tumor Cells

Methods for the characterization of not only tumor cells, but also rarecells, or other biological entities from biological samples have beenpreviously described (U.S. Pat. No. 6,365,362). This two stage methodrequires efficient enrichment to ensure acquisition of target cellswhile eliminating a substantial amount of debris and other interferingsubstances prior to analysis, allowing for cellular examination byimaging techniques. The method combines elements of immunomagneticenrichment with multi-parameter flow cytometry, microscopy andimmunocytochemical analysis in a uniquely automated way. The combinationmethod is used to enrich and enumerate epithelial cells in bloodsamples, thus providing a tool for measuring cancer.

The two stage method has applications in cancer prognosis and survivalfor patients with metastatic cancer (WO 04076643). Based on the presenceof morphologically intact circulating cancer cells in blood, this methodis able to correlate the presence of circulating cancer cells ofmetastatic breast cancer patients with time to disease progression andsurvival. More specifically, the presence of five (5) or morecirculating tumor cells per 7.5 milliliters provides a predictive valueat the first follow-up, thus providing an early prognostic indicator ofpatient survival.

The specificity of the assay described above increases with the numberof cells detected and is not sufficient in cases were only few(generally less than 5 circulating tumor cells) are detected. Onesolution to this problem is to provide detailed genetic informationabout suspected cancer cells. Accordingly, a method that wouldincorporate enrichment of a blood sample with multi-parametric imagecytometry and multi-parametric genetic analysis on an individual suspectcancer cell would provide a complete profile and confirmatory mechanismto significantly improve current procedures for patient screening,assessing recurrence of disease, or overall survival.

Fluorescent in situ hybridization (FISH) has been described as a singlemode of analysis in rare cell detection after enrichment as described inWO 00/60119; Meng at al PNAS 101 (25): 9393-9398 (2004); Fehm et al.Clin Can Res 8: 2073-2084 (2002) and incorporated by reference herein.After epithelial cell enrichment, captured cells are screened by knownhybridization methods and imaged on a microscope slide. Because ofinherent technical variations and a lack of satisfactory confirmation ofthe genetic information, the hybridization pattern alone does notprovide a level of clinical confidence that would be necessary forsensitive analysis, as in assessing samples with less than 5 targetcells. Further, this method for FISH analysis is difficult to automate.

Coupling hybridization-based methods with immunocytochemistry in theanalysis of individual cells has been previously described (U.S. Pat.No. 6,524,798). Simultaneous phenotypic and genotypic assessment ofindividual cells requires that the phenotypic characteristics remainstable after in situ hybridization preparatory steps and are limited inthe choice of detectable labels. Typically, conventional in situhybridization assays require the following steps: (1) denaturation withheat or alkali; (2) an optional step to reduce nonspecific binding; (3)hybridization of one or more nucleic acid probes to the target nucleicacid sequence; (4) removal of nucleic acid fragments not bound; and (5)detection of the hybridized probes. The reagents used to complete one ormore of these steps (i.e. methanol wash) will alter antigen recognitionin subsequent immunocytochemistry, cause small shifts in the position oftarget cells or completely removes the target cells, which introducesthe possibility of mischaracterization of suspect cells.

Probe sets and methods for multi-parametric FISH analysis has beendescribed in lung cancer (US 20030087248). A 3 probe combinationresulting in 95% sensitivity for detecting bladder cancer in patientshas also been described, see U.S. Pat. No. 6,376,188; U.S. Pat. No.6,174,681. These methods lack the specificity and sensitivity forassessing small numbers of target cells, and thus a confirmatoryassessment for early detection of disease state. They also do notprovide a means for convenient automation.

One aspect of the present invention provides a confirmatory assay in theanalysis of rare circulating cells by combining phenotypic and genotypicmultiparametic analysis of an individually isolated target cell,resulting in a clinically significant level of sensitivity and,therefore, assurance to the clinician of any quantitative informationacquired. Relevant disease states are assessed using extremely small (1,2, 3, or 4) numbers of circulating tumor cells (CTC's) and provide aconfirmation for early disease detection.

SUMMARY OF THE INVENTION Generation of Repeat Depleted DNA Probes

One embodiment of the present invention includes methods andcompositions to eliminate repetitive sequences from DNA. Any doublestranded DNA is a suitable source in the application of the methods ofthe present invention. To obtain single stranded DNA, devoid ofrepetitive sequences, first an amplified whole genome library is madefrom the source DNA according to standard procedures. The libraryobtained consists of randomly selected fragments ranging in size fromapproximately 200 to 500 base pairs. Each fragment consists of doublestranded DNA, having PCR primer sequences at each end of a targetsequence. Generally, this library is representative of the source DNA.Other methods that results in modified fragments of DNA to permitamplification are also considered in this invention with no limit to thesize of the fragments. These include, but are not limited to, degenerateoligonucleotide primed polymerase chain reaction (DOP PCR), rollingcircles and isothermal amplification methods. Double stranded DNAfragments are denatured by heating up to 95° C. or other means to obtainsingle stranded DNA fragments. The resulting single stranded DNAfragments contain repetitive sequences, unique sequences or acombination of unique and repetitive sequences. An excess of Cot DNA orother appropriate subtractor DNA that binds to repetitive sequences isadded. Subsequent lowering of the temperature results in the formationof double stranded DNA for only those fragments that contain repetitivesequences. Duplex Specific Nuclease (DSN) is added to allow digestion ofdouble stranded DNA. In one embodiment, the DSN enzyme is added for 2hours at 65° C. The resulting composition contains mostly singlestranded DNA, having only unique sequences, and digested DNA. The uniquesequence, now single stranded DNA with PCR primers at both ends, is usedas a template to generate large amounts of the unique sequence for usein probe production. When BAC clones containing a desired uniquesequence is used as source DNA, the template generated by this methodcontains only that unique sequence. When the boundary sequences areknown, this method is useful in obtaining probes that cover thenucleotides between the boundary sequences in genomic DNA. Further, thepresent invention includes methods of use and compositions, resultingfrom the production of these DNA sequences after elimination of theirrepetitive sequences. These repeat depleted DNA sequences function ashybridization probes without the use of a blocking DNA in anyappropriate application requiring disabling or blocking of undesired DNAsequences.

Another embodiment of the present invention provides for a system,apparatus, and methods in the preservation of immunomagnetically labeledcells for subsequent FISH analysis. This aspect permits the reanalysisof individual cells, utilizing the same or similar reporter moleculespreviously used to identify them. Accordingly after immunomagneticselection and initial fluorescent labeling, the cells of interest areidentified and their location is recorded. The cells are fixed inposition followed by appropriate processing. Alternatively, the cellsare fixed in position and stored for processing at a later point intime. For FISH applications, the sample is heated above the meltingtemperature of DNA, resulting in the loss of reporter molecules used toinitially identify the target cells. After completing FISH in which thefluorescent FISH probes are hybridized and the nuclear material is againfluorescently labeled, the sample is reintroduced in an analyzer whichlocates the cells of interest to examine fluorescent signals from theFISH probes.

Another embodiment of the present invention provides methods for thereanalysis of immunomagnetically labeled cells as a confirmation inidentifying rare circulating cells such as circulating tumor cells(CTC's). Thus, methods and techniques for the further processing ofcells after enrichment, immunofluorescent labeling and subsequentconfirmatory analysis, using in situ hybridization, as a means toincrease specificity and thereby confirm the identity of suspect CTC'sin patients as being cancer cells. Cytogenetic abnormalities detected inmorphologically suspect CTC's, detected in metastatic carcinomapatients, have a prognosis similar to patients with morphologicallyobvious CTCs or having an abundance of CTCs. One embodiment of thepresent invention considers confirmation assays in patients diagnosedwith carcinomas and having CTCs, or disseminated tumor cells (DTC's) inbone marrow, where there is an increased risk for recurrence. Inaddition, the methods of the present invention are applicable when thereis a need to assess for the presence or absence of drug targets in CTCsuch as, but not limited to, Her1, Her2, Androgen Receptor (AR), cMyc,or P10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation depicting the generation of repeatdepleted DNA probes from BAC starting DNA. A fragmented whole genomeamplification library is denatured and allowed to re-anneal in thepresence of excess Cot DNA. DSN digestion of the double strand DNAresults in a mixture of single strand unique sequence, available as atemplate for probe production.

FIG. 2: Schematic representation depicting the cleavage of a specificDNA sequence from a DNA source and production of clones thereof. Doublestranded DNA from an appropriate source is denatured and specific DNAsequence is allowed to hybridize. DSN digestion of the double strand DNAfollowed by separation of single strand DNA by size results in isolationof the desired single strand DNA. After synthesis of the second strand,the desired DNA is cloned into an appropriate vector for production.

FIG. 3: Comparison using repeat-depleted probes in FISH analysis. Whiteblood cells hybridized with a Her-2 probe containing repeats and noblocking DNA. In the absence of blocking DNA, the probe labels theentire nucleus after hybridization with repeat regions. Panel A showswhite blood cells hybridized with a Her-2 FISH probe containing repeatsand no blocking DNA. Panel B shows the same cells after labeling thenucleus with DAPI. Panel C shows the overlay of the two signals and thelack of Her-2 resolution. Panel D shows FISH analysis on white bloodcells using repeat-depleted a Her-2 probe. Arrows indicate locations ofunique chromosome sequence for Her-2. Panel B shows the same cells afterlabeling the nucleus with DAPI. Panel F shows the overlay of panel D andB, visualizing the location of the Her-2 site within the cell nucleus.

FIG. 4: Panel A shows a chromosome spread hybridized with a P16 (CDKN2A)labeled repeat free probe targeting 9p21 frequently used to characterizemelanoma. Two chromosomes show the presence of 9p21 and are illustratedby arrows. Panel B shows a chromosome spread hybridized with MLL labeledrepeat free probe targeting 11q23 used to identify a specific type ofleukemia. Two chromosomes show the presence of 11 q23 as illustrated byarrows.

FIG. 5: Schematic representation of the fixation and hybridizationdevice used to prepare samples for FISH analysis. A compact and portabledevice for preparing a sample for FISH analysis after immunomagneticenrichment and initial fluorescent imaging. Shown are the control panel,electronics, pump, and poser supply in relation to the sample cartridge.

FIG. 6: Schematic representation of the basic steps for FISH afterinitial fluorescent imaging. Shown are cross-sectional images of thesample cartridge and the presence of magnetic support (black wedges).Panel 1 shows the cells arranged along the internal surface of theimaging face of the cartridge after initial fluorescent imaging usingCellTracks System. Panel 2 shows the simultaneous replacement of thebuffer solution with a fixative for FISH. In Panel 3, the fixative isaspirated to remove fluids from the cartridge. Panel 4 shows theaddition of forced air to dry the cartridge. In Panel 5, the cartridgeis inverted and enough FISH probe is added to cover the cells. Panel 6shows the cartridge on a heat source to allow hybridization. In Panel 7,the FISH reagents are washed to allow rescanning and analysis of theFISH signals in Panel 8.

FIG. 7: Schematic representation of the stopper with probe extension forFISH cartridge for reducing the chamber volume of the cartridge.

FIG. 8: Schematic representation of the cartridge. A cross-sectionalillustration depicts the cartridge inverted with the location of thecells and FISH reagents illustrating a low reagent volume distributionthe lower face of the chamber, thus allowing enough reagents to onlycover the cells along entire lower surface.

FIG. 9: Representative image of a tumor cell initially identifiedthrough immunocytochemistry (ICC) with subsequent FISH analysis for thepresence of chromosome 1, 7, 8 and 17. Panel A shows a list of CTCcandidates identified by the software on basis of their ICC signature.Panel B shows the acquired fluorescent ICC images acquired. Panel Cshows the corresponding FISH signals for chromosomes 1, 7, 8 and 17demonstrating the aneuploid signature of the same cell tumor cell.

FIG. 10: Shown are five fluorescence images at different focal planesthrough the cell using excitation/emission filters for 5 differentfluorochromes. Panel A shows the images for a cell using PE. Panel Bshows images for the same cell using DAPI. Panel C shows images for thesame cell using APC. Panel D shows images for the same cell using FITC.Panel E shows images for the same cell using Dy415.

FIG. 11: Results from ICC and FISH analysis to confirm a CTC. Panel Ashows ICC images of the ICC scan on which a suspect CTC was identified.Panel B shows the corresponding fluorescence signals, using FISH probes.Corresponding counts of the signals for each probe are shown next toeach image; 4 count for PE, 1 count for APC, 4 count for FITC and 2count for Dy415.

DETAILED DESCRIPTION OF THE INVENTION Generation of Repeat Depleted DNAProbes

DNA contains unique as well as repetitive sequences. The repetitivesequences occur throughout the chromosomes and have the potential tointerfere with hybridization reactions, such as with in situhybridization, targeted toward specific regions or unique sequencesoutside these repetitive sequences. To identify the presence, amount andlocation of specific sequences on chromosomes, genes or DNA sequences itis important that the hybridization probes hybridize only at thelocation of interest. The presence of repetitive sequences in thehybridization probe mixture reduces the specificity of the binding,requiring methods to either remove the repetitive sequences from theprobes or prevent the probes from hybridizing to the repetitivesequences on the target. For example, Cot-1 DNA is often added duringhybridization to prevent binding of the probes to the repetitivesequences (U.S. Pat. No. 5,447,841 and U.S. Pat. No. 6,596,479).

Recent contributions have addressed this question by disabling therepetitive sequences. The use of Cot-1 DNA relies on the ability ofCot-1 DNA to form a duplex structure with available single strand repeatsequences, and thereby minimize non-specific binding interaction of thisportion of the sequence with the unique target sequence. Blocking therepetitive DNA, either during a hybridization step with the uniquetarget sequence or prior as in a pre-association step, results in amixture having repetitive segments forming duplex structures with theircomplementary sequence and a single strand form of the target probe,available for hybridization to its unique target segment. Unfortunately,the presence of this duplex in a subsequent amplification or labelingreaction affects the signal through the introduction of non-specificnoise, especially in situations where the signal is very weak. Analternative to blocking the repetitive sequence is to remove theunwanted repeat segments from the reaction mix.

Generation of repeat-depleted DNA probes of this prevention is depictedin FIG. 1. One embodiment of the present invention makes use of duplexspecific nucleases (DSN) which preferentially cleave deoxyribonucleicacid molecules (US 2005/0164216 and U.S. Pat. No. 6,541,204 incorporatedby reference). The ability of the enzyme to preferentially cleavenucleic acid duplex polynucleotides as compared to single strand DNAprovides a means for removing non-target double stranded DNA from thesample mixture. The ability of these nucleases to preferentially digestthe duplex form of polynucleotides provides potential use inmanufacturing an unique target specific probe, eliminating theinterfering affect of blocking DNA, and providing a means for theirrapid, efficient and cost effective production.

Starting DNA used in the practice of this invention is typically in theform of one or more DNA sequences which contain a multiplicity of DNAsegments. The initial source of individual starting material in theproduction of the probe composition has been described in the productionof direct-labeled probes (U.S. Pat. No. 6,569,626). Optimally the sourceof the starting polynucleotide is purified from tissue and fragmentedinto 150 kb to 200 kb segments, using any known technique such as, butnot limited to, enzyme treatment (restriction enzymes), polymerase,limited DNase I digestion, limited mung bean nuclease digestion,sonication, shearing of DNA, and the like. Some of these segmentalfragments will be complementary to at least a portion of one or more DNAsegments in the particular unique target sequence. The individual DNAsegments are propagated by commonly known methods, such as cloning intoa plasmid construct and then transfected into bacteria. Afterpropagating the cloned fragments, individual colonies representingisolated fragments are identified as containing at least a portion ofthe sequence of interest. Identification is accomplished by knowntechniques such as hybridization, PCR, or searching establisheddatabases of commercially available libraries. Each chosen colony isgrown to obtain an isolated plasmid construct having a unique fragment,at least partially complementary to a segment of the target sequence onthe chromosome. Exemplary target sequences include HER-2, IGF-1, MUC-1,EGFR, and AR and may be available through commercial vendors (i.e. BACclones). Once the cloned fragments of interest are propagated andisolated, they are depleted of their repetitive polynucleotidesequences. Using whole gene amplification (WGA), the fragments areamplified as 200 to 500 bp segments from the isolated plasmidconstructs. Commercially available DOP PCR is considered as oneembodiment to this portion of the procedure. Cot-1 DNA is combined withthe WGA library pool after amplification by first heating to 95° C. todenature the double-strand polynucleotide into a single strand state andthen cooling to 65° C. to allow selective re-annealing of the repeatsequences. Duplex specific nucleases (DSN) under optimized DSNconditions are then added to preferentially cleave deoxyribonucleic acidmolecules containing perfectly matched nucleic acid duplexes while notaffecting any remaining single stranded segments. Selectively cleavingthe duplex nucleic acids is accomplished by enzymatic digestion ofDNA-DNA duplexes and DNA-RNA duplexes. Specific embodiments of thepresent invention include DSN isolated from the Kamchatka crab (U.S.Ser. No. 10/845,366) or shrimp (U.S. Pat. No. 6,541,204), but anyenzymatic removal of duplex structure is considered in the presentinvention. The use of endonuclease-specific nucleases hydrolyzes aphosphodiester bond in the duplex DNA backbone, providing the advantageof not being nucleotide sequence-specific and therefore applicable tomost targets of interest. DSN digestion provides for the removal of asubstantial amount of the nucleic acid duplex for subsequentamplification of the remaining single-strand polynucleotide. Oneembodiment of the present invention is a 2 hour DSN digestion at 65° C.The resulting composition contains single stranded DNA, corresponding toportions of the unique target sequence on the chromosome, some amount ofundigested double-strand DNA, and digested base pairs. Preferably, theundigested DNA is separated from the digested DNA and the DSN bycentrifugation (i.e. spin column chromatography). The mixture is usedimmediately or stored at 80° C., either before or after amplification ofthe purified composition for subsequent utilization such as labeling anduse for in situ hybridization. After amplification, the resulting targetprobe sequence is amplified by PCR yielding 90% to 99% pure target probesequence, and designated repeat-depleted DNA.

The use of DSN in the enrichment and isolation of a single strandpolynucleotide from double strand is applicable in the production of anysingle strand polynucleotide wherein separation of the single strandentity from double strand contaminants is desirable. This isparticularly relevant, although not limited, in the production oflabeled probes for gene or chromosome identification, karyotype orpanning a pool of single strand and double strand polynucleotides.

The resulting probes, both composition and production, are incorporatedin the subject matter embodied in the present invention. Repeat-depletedDNA, as described in the present invention, is useful for in situhybridization, including FISH, and all other nucleic acid hybridizationassays. The requirement for competitive binding is eliminated using therepeat-depleted probes described in this invention, resulting inincreased specificity of the reaction and a reduction in the amount ofprobes necessary for binding.

The Duplex Specific Nuclease Method

To make a hybridization probe toward a target sequence, DNA containingthe sequence of interest is obtained. Methods to obtain DNA containingsequences of interest will be known to those skilled in the art andinclude, without limitation, isolation of genomic DNA from tissues orcells, flow sorting of chromosomes, and screening libraries of clonedfragments of chromosomes by hybridization, electronically, or PCR.

Starting DNA used in the practice of this invention is purified from asource by any method. Typically the starting DNA consists of genomes,chromosomes, portions of chromosomes, or cloned fragments ofchromosomes. Flow-sorted chromosomes and Bacterial ArtificialChromosomes (BAC) known to contain target sequences of cancer relatedgenes make the present invention particularly applicable. Exemplarytarget sequences include HER-2, IGF-1, MYC, EGFR, and AR. BAC clonescontaining these sequences are available through commercial vendors.

Once the DNA containing sequences of interest are identified andobtained, they are depleted of repetitive polynucleotide sequences. Thisprocess begins by fragmenting and preparing a library containing thesequence of interest. One method is the GenomePlex® Whole GenomeAmplification (WGA) method (GenomePlex® is a trademark of RubiconGenomics, Inc.) that randomly cleaves the cloned fragments into 200-500bp fragments and attaches linker sequences which can then be used toamplify and re-amplify the library using PCR. In this example thefragmented, amplified library is considered the source DNA.

To remove the repetitive sequences, the source DNA is denatured to asingle stranded state and then cooled under conditions that selectivelyallow repetitive sequences to anneal to form double stranded moleculesand unique sequences to remain single stranded. A duplex specificnuclease (DSN) is then added which preferentially cleaves the doublestranded repetitive fragments while not cleaving the single strandedunique sequences. The resulting mixture contains single stranded DNA,corresponding to portions of the unique target sequence on thechromosome, some amount of undigested double-strand DNA, and digestedbase pairs. Preferably, the undigested DNA is separated from thedigested DNA and the DSN by spin column chromatography, phenolchloroform extraction or some other similar method, but separation isnot a requirement. Then, the repeat-depleted library is used as ahybridization probe or re-amplified using PCR to prepare larger amountsof probe DNA. After amplification, the resulting target probe sequenceis 90% to 99% pure target probe sequence, and designated Repeat-depletedDNA.

The library fragmentation and amplification methods described above arenot intended to be limiting but rather serve as an example of how onefragmentation and amplification method is used to make repeat-depletedprobes. There are numerous methods of fragmenting and amplifying nucleicacids including linker-adapter PCR, DOP PCR, rolling circleamplification, transcription-mediated amplification and all othermethods are considered this invention. It is expected that somemodification of the above method to prepare repeat-depleted DNA will benecessary to accommodate the different methods of library fragmentationand amplification and these modifications are also included in thepresent invention.

One consideration in this invention is the use of an enzyme that iscapable of cleaving double stranded DNA while not cleaving singlestranded DNA. Enzymes included in this invention may cleave doublestranded DNA in any way, including lysis of the sugar-phosphatebackbone, removal of one or both strands in a DNA duplex or removal ofnitrogenous bases to form apurinic/apyrimidinic sites. Non-limitingexamples of these enzymes include endonucleases, exonucleases,restriction enzymes, nicking enzymes, DNA repair enzymes,topoisomerases, DNA gyrases, and enzymes involved in homologousrecombination. Specific embodiments of the present invention include DSNisolated from the Kamchatka crab (U.S. Ser. No. 10/845,366), shrimp(U.S. Pat. No. 6,541,204), T7 Endonuclease I, and E. coli exonucleaseIII, all incorporated by reference.

Enzyme concentration, time of digestion, and buffer conditions such assalt and magnesium ion concentration are factors that can affect thespecificity of DSN toward double stranded DNA. Optimization of theseconditions is necessary to get efficient repeat-depletion.

Efficiency and specificity of repeat removal in this invention aredependent on the reaction conditions used to denature and re-anneal aswell as the conditions present during digestion with the DSN.Denaturation is accomplished by alkali or heating. The degree of DNAre-annealing is dependent on the concentration of DNA present in thesamples and the time allowed for re-annealing. In order for selectivere-annealing of repetitive sequences to occur, the repeat sequences mustbe present in a higher concentration than the unique sequences. Theratio of repeat to unique sequences within a particular clone will varyfrom region to region throughout the genome. To standardize thedepletion process across regions with varying numbers of repeatsequences, an excess of subtractor DNA is added to the reaction. Themass of subtractor DNA added varies depending on the desired amount ofrepeat removal and is preferably 10-50 times the mass of the source DNA.The present invention considers that a subtractor is any nucleic acid ornucleic acid analogue containing sequences sufficiently homologous innucleotide sequence to the repetitive sequences as to allowhybridization between subtractor sequences and a portion of thesequences in the source DNA, making the subtractor sequence useful. Oneembodiment of the present invention includes Cot-1 DNA as a subtractorDNA which is used to remove repetitive sequences from source DNA.

The stringency for re-annealing is another component in the presentinvention. Salt concentration and temperature are factors that determinethe stringency of any re-annealing step. The degree of repeat removal iscontrolled by adjusting stringency conditions for this step. Adjustingthe stringency conditions to allow some degree of annealing betweensequences that are not 100% homologous improves the degree of repeatremoval. Salt concentrations range from 5 millimolar to 1000 millimolarNaCl with annealing temperatures range from 15° C. to 80° C.

In one embodiment, the repeat-depletion process is performed such thatre-annealing and DSN digestion occur sequentially. Accordingly, the DNAis denatured and allowed to cool for a period of time under conditionsoptimized for annealing. Then, the reaction conditions are changed toconditions that optimize the specificity and activity of DSN digestion.In another embodiment, the re-annealing and DSN digestion take placesimultaneously, under the same conditions.

Also within the scope of this invention, the source or subtractor DNA istreated with an agent, either chemical or physical, before or duringenzymatic digestion to alter the specificity of an enzyme toward eitherthe single stranded or double stranded fractions within the mixture. Forexample, E. coli RecA protein is added to a mixture of single strandedand double stranded DNA. This protein coats the single stranded DNA inthe mix and protects the single strand DNA from E. coli RecBC DNasewhile allowing the double stranded DNA in the mixture to be digested(“Echerichia coli RecA protein protects singles stranded DNA or GappedDuplex DNA from degradation by RecBC DNase”. Williams, J G K, Shibata,T. Radding, CM Journal of Biological Chemistry V246 no. 14 pp7573-7582). It is also possible to generate source or subtractor DNAusing modified nucleotides which alter the specificity of an enzymetoward the single strand or double strand DNA fractions.

The use of DSN in the enrichment and isolation of a single strandpolynucleotide from double strand is applicable in the production of anysingle strand polynucleotide wherein separation of the single strandentity from double strand contaminants is desirable. This includesremoval of any undesirable sequence from a source DNA. These undesirablesequences include without limitation, repetitive sequences, uniquesequences, and vector sequences. This method is particularly relevant inthe production of labeled probes for gene or chromosome identification,karyotyping, or panning a pool of single strand and double strandpolynucleotides.

The Selective Binding Method

By denaturing source DNA and selectively allowing repetitive sequencesto anneal, any agent that binds preferentially to a single or doublestranded DNA structure is used to remove repeat sequences from sourceDNA. Examples of these agents include, without limitation, DNA or RNApolynucleotides, enzymes, antibodies, DNA binding proteins, combinationsof antibodies and DNA binding agents, and natural or synthetic compoundsand molecules. DNA binding agents may be linked directly or indirectlyto a solid support which allows for positive or negative chromatographicselection of unique or repetitive sequences. One example includesseparation of single and double strand DNA using biotinylated antibodiestoward single strand or double strand DNA. The desired population isseparated using streptavidin-coated paramagnetic particles. Alternately,a biotinylated antibody toward a DNA binding agent that preferentiallybinds single or double strand DNA is used in the same fashion.

Other Single or Double Strand Specific Enzymes

The present invention also embodies any enzyme that preferentially actson single or double strand DNA in modifying source or subtractor DNA infacilitating repeat removal One non-limiting example is to selectivelyligate a DNA linker to the double stranded DNA population afterdenaturation and selective re-annealing the repeats. This linker ishybridized to a homologous oligonucleotide attached to a magnetic (orparamagnetic) particle to remove repetitive sequences. A second exampleis to use a single strand DNA/RNA ligase to selectively circularizesingle stranded DNA present after denaturation and selectivere-annealing the source DNA. The resulting circles are then be amplifiedand enriched by rolling circle amplification.

Structure Specific Separation of Repetitive Sequences

In addition to specific probe production methods and based on separationof single stranded DNA from double strand DNA, the present inventionconsiders any method known in the art whereby separation of repeatsequences from unique sequences occurs with the establishment somedetectable DNA structure in either the repeat sequences or the uniquesequences and this detectable structure is used to separate onepopulation from the other. Some examples of detectable DNA structuresinclude without limitation, triple or quadruple stranded DNA, hairpins,panhandles, flaps, Z-DNA, Holliday junctions and other structures formedduring recombination. These structures may be naturally occurring withinthe sequences of interest or they may be induced by modifying either orboth the source nucleic acid or the subtractor nucleic acid.

Selective Digestion of Repeat Sequences

Another method to remove repeat sequences from source DNA is to digest afragmented and amplifiable DNA library with a restriction enzyme whoserecognition sequence is known to exist only in repetitive DNA sequences.When the digested source DNA is re-amplified by PCR, the remaininglibrary will be enriched for unique sequences and depleted of sequencesthat contain repeats.

Digestion and Selective Ligation

Another method is to prepare repeat-depleted probes is to digest targetDNA with two restriction enzymes that leave different overhangs on thedigested sequence. The first restriction enzyme is preferably an enzymethat cuts within the repeat sequences and the second is an enzyme thatthe does not cut within the repeat sequences. Following digestion,linkers are selectively attached to the ends of the sequences cut by thesecond restriction enzyme. These linker sequences are then used to PCRamplify a library, depleted of repeat sequences. The resultingrepeat-depleted DNA, both composition and production, are incorporatedin the present invention. Repeat-depleted DNA, as described in thepresent invention, is useful as probes for any type of hybridizationassay where specific binding of target sequences is desired. Thesetechniques include, without limitation, ISH, FISH, CGH, spectralkaryotyping, chromosome painting, Southern blot, Northern blot, andmicroarrays. Production of hybridization probes that only contain uniquesequence is one embodiment of the present invention. Consequently, therequirement for competitive binding is eliminated, resulting in anincrease in the specificity of the reaction, and reducing the amount ofprobes necessary for binding.

Use of Duplex Specific Nuclease to Cleave DNA at a Desired Location.

The use of duplex specific nucleases has utility in cleaving specificsequences from DNA. avoiding the use of BAC clones, and increasing thespecificity of the DNA probes. FIG. 2 show a schematic representation ofthis embodiment Single strand DNA is obtained from a DNA source,containing the desired sequence. Primers identifying both ends of thedesired sequence are added, and duplex specific nucleases introduced tocut the desired DNA probe from the source DNA. After separating thedesired DNA probe by size exclusion, second strand DNA is synthesizedand cloned to provide a source for DNA probes. Thus, duplex specificnuclease are used to cleave DNA sequences at specific regions. Thismethod is most useful in isolation of fragments of interest when thefragments lack appropriate restriction enzyme sites. In FIG. 2, twooligonucleotides are designed to anneal to one or both strands of theDNA, and flank the sequence of interest DNA containing a sequence ofinterest is denatured and allowed to re-anneal in the presence of anexcess of the flanking oligonucleotides. Digestion with a duplexspecific nuclease selectively cuts the DNA at the site where theoligonucleotides are annealed. The remaining single strand DNA moleculesare fractionated by size to obtain the sequence of interest. Thesequence of interest is made double stranded using a DNA polymerase andcloned into a plasmid. Thus making possibilities to clone or subclonesequences of interest from a larger DNA polynucleotides. A secondexample of site specific cleavage is the recovery of cloned fragmentsfrom plasmid vectors by selectively digesting the vectors. In oneexample, the plasmid containing cloned DNA fragment is denatured andallowed to re-anneal in the presence of excess plasmid, lacking clonedDNA. Addition of a duplex specific nuclease cleaves the plasmidsequences and leaves the single strand cloned DNA intact. The remainingsingle strand DNA is then be used for any application known in the artincluding, but not limited to, sequencing or subcloning.

Example 1-Detection of Chromosomes or Portions of Chromosomes UsingRepeat-Depleted DNA Probes

BAC clone CTD-2019C10 was selected to be used as a probe for the Her-2gene by electronically screening the human genome using the UCSC GenomeBrowser software (http://genome.ucsc.edu/cgi-bin/hgGateway) and cloneswere obtained from Invitrogen (Carlsbad, Calif.). BAC DNA was isolatedusing the Large Construct Kit from Qiagen (Valencia, Calif.). Source DNAwas prepared using 10 nanograms of purified BAC and the Genomeplex®Complete Whole Genome Amplification Kit (Sigma-Aldrich St. Louis, Mo.)according to the manufacturer's directions. Depletion mixes wereprepared containing 2 micrograms of Cot-1 DNA, 1× Duplex SpecificNuclease buffer (Evrogen, Moscow, Russia), 03 molar NaCl, and 66nanograms of source DNA. The depletion mixes were denatured for minutesat 95° C., placed on ice for 10 seconds and 1 unit of Duplex SpecificNuclease (Evrogen, Moscow, Russia) added. Samples were incubated at 65°C. for 90 minutes. Five microliters of the reaction were purified usingthe Genelute PCR Clean-Up Kit (Sigma-Aldrich St. Louis, Mo.) and thepurified DNA was eluted in 50 microliter aliquots. Fifteen microlitersof the depleted samples were then re-amplified by PCR using the WholeGenome Re-amplification kit (Sigma-Aldrich St Louis, Mo.). PCR reactionswere purified as described and quantified based upon their A₂₆₀. Tennanograms of the first re-amplification mixture was used as template ina second re-amplification, purified as described. This material wassonicated to an average molecular weight of 200-500 base pairs, ethanolprecipitated, and resuspended in distilled H₂O. The resulting DNA wasfluorescently labeled using the Kreatech ULS Platinum Bright Red/OrangeKit (Kreatech, Amsterdam, Netherlands). For comparison, probes withrepeats were also made from the source DNA which was used in thedepletion process.

Phytohemagglutinin-stimulated white blood cells were prepared for FISHby fixation in 75% methanol, 25% acetic acid and spotted on slides usingstandard techniques. Repeat-depleted probes and source DNA probes werehybridized at 2 ng/μl without Cot blocking DNA in a hybridization bufferconsisting of 50% formamide, 10% dextran sulfate, and 1×SSC. Slides andprobe were co-denatured at 80° C. for 3 minutes and hybridized overnightat 37° C. Following hybridization, samples were washed for five minutesat 50° C. in 0.5×SSC, 0.001% SDS. Samples were counterstained in 0.5ng/ml DAPI for 5 minutes and mounted in 50% glycerol. Images wereacquired using a Leica DM-RXA fluorescent microscope (LeicaMicrosystems, Bannockburn, Ill.) equipped with filters appropriate forrhodamine and DAPI. Images were acquired with a Photometrics SynSysblack and white digital camera (Photometrics, Tucson, Ariz.). DAPIsignals were enhanced and overlay images were generated using Leica FW4000 software. Her-2 images are unedited and were captured usingidentical camera settings comparison purposes. FIG. 3 depicts acomparison of the images. Panel A shows that when source DNA containingrepeats is used as a hybridization probe, the probes stain the entirenucleus and no Her-2 specific signals are visible. When repeat-depletedDNA is used as a probe (Panel D) specific signals that correspond to theHer-2 gene are clearly detectable (arrows).

Example 2—DNA Probes Depleted from Repeat Sequences According to thisInvention Improves the Visualization of Fluorescently Labeled DNA Probesas Compared to Traditional DNA Probes that Contain Repeats which areBlocked During the Procedure

The signal to noise ration of fluorescently labeled probes issignificantly improved when employing repeat depleted DNA probesobtained according to the invention. For this comparison, DNA probestargeting 9p21 and 11q23 were used as they are known to those skilled inthe art. These signals are problematic in that they are relatively smallsignals and difficult to discern. Probes were depleted from repeatsequences according to the invention and fluorescent reporter moleculelinked to a platinum group which forms a coordinative bond t the N7position of guanine was used to fluorescently label the probes (ULSlabeling, Kreatech, Amsterdam). FIG. 4 Panels A and B show a chromosomespread hybridized with rhodamine labeled 9p21 and dGreen labeled 11q23probe respectively. Clear signals from the repeat free probes can bediscerned with the repeat free probes as indicated by arrows in thefigures. Thus, the visualization of the presence of these probes issuperior to those that are obtained using probes that are obtainedthrough traditional methods. This improvement in visualization providesa more accurate differential diagnosis of melanoma (Panel A, 9p21, P16(CDKN2A) and leukemia (Panel B, 11q23, MLL).

Preservation of Immunomagnetically-Labeled Cells for Subsequent Analysis

During immunocytochemistry (ICC) image analysis the cells aremagnetically held to the optically transparent surface of the cartridgeby magnetic forces applied by an external magnet (U.S. Pat. No.5,466,574). The calculated holding force of the device is approximately10⁻⁹ Newtons. This holding force is dependant upon several variablesincluding but not limited to the number of ferrofluid particles on thecell, the size of the magnetic particles and the magnetic field gradientapplied by the external magnet. In order to fix the cells to the glasssurface, the buffer solution must be removed and replaced by a cellfixative solution, such as methanol, acetone, acetic acid, other agentsknown in the art and combinations of these. Aspiration of the buffersolution must be carefully completed so as to not displace or remove thecells to be analyzed. So as fluid is aspirated from the sample chamber,the meniscus of the fluid applies shear forces on the magnetically heldcells. These shear forces can be greater than the magnetic holdingforces (calculated at greater than 10⁻⁹ Newtons). In such situations,the cells will either be moved within the cartridge or displaced suchthat they are aspirated from the cartridge along with the buffersolution. Fluid shear forces are a function of the rate at which themeniscus moves across the glass portion of the cartridge, the distancebetween the aspiration probe and the glass surface, the velocity andviscosity of the fluid being aspirated and other parameters.Additionally after aspiration, any drying of the glass surface beforethe fixation solution is added can have a negative effect on the cellswithin the cartridge. Plus, the addition of a fixation solution into anempty cartridge will further disturb the distribution of the cells.

Accordingly, one aspect of the present invention address these issues byproviding a method for replacing the buffer solution with the fixationfluid without subjecting the cells to fluid shear forces caused by themeniscus. Fixation solution is dispensed into the bottom of thecartridge with the simultaneous aspiration of the displaced buffersolution from the top of the cartridge. While some mixing of fixativeand buffer will take place at the interface of the two fluids,sufficient fixation solution will be dispensed to complete the requiredcell fixation to the glass surface. This fluid displacement will occurwith minimal shear forces applied to the cells in the cartridge bybalancing the flow between the dispensed fixative solution andaspiration of the displaced fluid, in addition to the magnetic holdingforce retaining the immunomagnetic attached cells to the surface of theglass. One preferred embodiment of the present invention utilizes theentry area of sample chambers described in U.S. Pat. No. 6,861,259; U.S.Ser. No. 10/988,057; and U.S. Pat. No. 7,011,794; U.S. Ser. No.11/294,012 in displacing approximately 100 microliters of fluid withinthe cartridge without spilling out of the cartridge. The opening port ofthe cartridge is sufficient to allow an aspiration probe to removebuffer solution as the fixative solution is being dispensed to displacethe buffer solution. Once the cells have been fixed in place by thefixation fluid, the fluid may be removed without risk of celldisturbance. This procedure allows for automated processing of samplesfor subsequent FISH or other analysis with minimal operator interactionthat could introduce variability into the preparation process.

The present invention describes an automated device which allows forcomplete and consistent fixation of cells in the cartridge after ICCimaging in a bench-top device, and incorporates all the steps in thepreparation of target cells after ICC for subsequent FISH imageanalysis. FIG. 5 depicts a schematic view of the apparatus showing therelative locations of the individual components. Accordingly, thecartridge containing the ICC imaged sample is placed into the device forbuffer removal and fixation. A syringe and syringe pump in combinationwith a pipette aspirates the buffer and dispenses the fixative. FIG. 6is a schematic representation of the steps involved in the fixation andhybridization of the cells. In one embodiment of the invention thebuffer removal and addition of fixative are performed simultaneously tominimize cell movement through the forces exhibited by the fluid removaland addition. The fixation is completed by removal of all fluids fromthe cartridge followed by drying of the cartridge by a forced air flowinside the cartridge using the same pipette as used for the addition andremoval of fixation reagents. After fixation and drying the cartridge isstored or used immediately for FISH or other additional analysis.Optimal mixtures for the fixative differ depending on the target entity(i.e. DNA, RNA, protein).

Fixation Protocol for FISH

To fix the cells on the upper surface and leave them intact andaccessible for FISH probes, the following protocol is developed andimplemented in the automated bench-top device:

-   -   1. Dispense 250 microliters of fixative from the bottom of the        cartridge (cartridge in up-right position).    -   2. Aspirate 250 microliters from the top and dispose.    -   3. Repeat the dispense 250 microliters new fixative from the        bottom of the cartridge.    -   4. Aspirate all fluid from the top of the cartridge.    -   5. Dry the cartridge by flowing air through the cartridge.        Pipette used for aspiration/dispensing is used for air flow as        well.

Volume Reduction

As a consequence of the expense of antibodies or polynucleotide probesand the requirement to use them in high concentrations, reactions arecarried out in very small closed volumes (for example 5 microliters to25 microliters) so the cost of using a high concentration is offset byhaving to use very small volumes of reagents. Sample cartridges asdescribed in U.S. Pat. No. 6,861,259; Ser. No. 10/988,057; and U.S. Pat.No. 7,011,794; Ser. No. 11/294,012 are used as the reaction vessel afterimmobilization in the chamber. In these cartridges, the immediate volumeof the chamber where the cells are immobilized is 320 microliters. Thus,there is a need to analyze immobilized cells by in situ hybridization,but the adding 320 microliters of a high concentration of most probesare expensive and impractical.

To address this problem, a uniform distribution of the probe mixtureacross the surface of the optically transparent surface of the cartridgewhere the cells are immobilized is needed, while reducing the volume ofthe added probe. This method is obtained by the following:

-   -   1. Inserting an object inside the cartridge to reduce the        volume.    -   2. Using a volume that is large enough, but smaller that the 320        microliters across the entire surface where the cells are        immobilized when the cartridge is in a horizontal position and        the surface with the cells is downside (optical viewing surface        on bottom).    -   3. Use a small volume plus a fluid with a density that is lower        tan the density of the reagents. The low density fluid floats on        top of the reagent and allows the reagents to spread uniformly        across the entire surface.

As an example of the first possibility, an extension is introduced atthe probe end of the stopper so that a portion extends the full lengthof the chamber (FIG. 7). The extension consumes approximately ⅓ of thevolume of the chamber. The extension diameter is dimensioned such thatit will slide through the chamber opening, 2.36 mm diameter. Theextension is made by molding an entire new plug over the molding on theexisting plug, or inserting a solid metal rod through the center line ofthe plug. The material must be inert to the reagents as, for example,316 stainless steel, polypropylene or Inconel 625. The over molding ofthe upper portion of the plug with a thermal plastic is necessary toensure the proper plastic durometer for maintaining shape duringinsertion and when positioned in the chamber to maintain liquid seal andlocking of the plug. Further, the plug and extension optionally has anaccess hole through the center for monitoring temperature within thechamber during processing. The plug is further designed to be removedand re-used after proper cleaning.

A second embodiment is depicted in FIG. 8. A volume of 50 microliters ofFISH reagents is sufficient to cover the whole upper surface of thecartridge. This volume needed to ensure complete reactions and isdependent on the viscosity and hydrophobicity of the reagents. Afteraddition of the reagents, the cartridge is placed in a horizontalposition to further ensure exposure to the reagents.

One other embodiment incorporates the second embodiment with a furtherreduction in reagent volume. After injecting of the reagents asdescribed, an extra fluid with a lower density is injected. As the lowerdensity fluid floats on top of the reagents of the reagents, there is amore complete reaction over the entire surface. Depending on thecomponents of the reagents, a volume of 25 microliters reagents isobtained.

Reanalysis of Immunomagnetically-Labeled Cells

Chromosomal aneuploidy is associated with genetic disorders,particularly cancer. Diagnostic methods are available that provide forthe detection of these chromosomal abnormalities particularly with theuse of in hybridization (ISH). The application of ISH andimmunocytochemistry (ICC) on tissue or cell samples has been wellestablished, but there is a clear need to establish a diagnosticallyeffective method for the simultaneous analysis of ISH and ICC on asingle cell. One aspect of the present invention provides for thedetection of these chromosomal abnormalities on individual cells as theyrelate to the confirmation of morphologically suspect cancer cellsthrough a cost effective and highly specific means.

One aspect of the present invention provides for the further processingof rare cells after enrichment and immunocytochemical (ICC) analysis.For example, circulating rare cells such as epithelial cells areidentified as suspect cancer cells (U.S. Pat. No. 6,365,362; U.S. Pat.No. 6,645,731; and U.S. Ser. No. 11/202,875 are incorporated byreference). Suspect cells are identified through specific cellularantigens and nucleic acid labeling. Confirmation of these suspect cellsare subsequently determined by the expression of specific unique targetsequences, defining either a chromosome and/or gene, used to assesschromosomal changes (i.e. aneuploidy) within the identified suspectcell. Accordingly, one embodiment of the present invention includes thecombination of ICC staining and subsequent confirmation by fluorescentin situ hybridization (FISH) on a group of selected chromosomes whichdefine a CTC.

The cancer confirmatory assay provides an increased specificity afterimmunomagnetic enrichment and fluorescent imaging of circulating tumorcells as provided by the CellTracks® AutoPrep® and CellTracks® AnalyzerII Systems (Immunicon Corporation) and further described in U.S. Pat.No. 6,365,362. A confirmatory test permits the designation of 1 or moreCTC's as a cancer cell regardless of the stage of the disease and thuslowers the threshold for calling a sample positive for CTCs. Oneembodiment of the present invention is assessing aneuploidy inchromosomes 1, 7, 8 and/or 17 to confirm ICC-determined suspect CTC's. Afurther embodiment includes the detection of individual genes such as,but not limited to, HER-2, IGF-1, MYC, EGFR, and the androgen receptor(AR) to detect the presence or absence of therapeutic targets and thusprovides a means to make the correct choice of treatment.

Accordingly, an automated and standardized method for blood sampleprocessing provides identification of circulating epithelial cells byICC. Aspirated plasma from a partitioned blood sample is combined with aferrofluid reagent conjugated to antibodies specific for a target cellpopulation (i.e. EpCAM positive). These cells are immunomagneticallycollected through an externally applied magnetic field, allowing forseparation and removal of unlabeled cells.

Once the target cells are separated, they are dispensed into adisposable cartridge for image analysis using an image presentationdevice (U.S. Pat. No. 6,790,366 and U.S. Pat. No. 6,890,426). The deviceis designed to exert a magnetic field that orients the labeled cellsalong the optically transparent surface of the chamber for subsequentICC imaging.

After ICC imaging, suspect cells are identified using appropriatealgorithms. Images of the suspected cells are presented to the user whomakes the final decision about the identity of the presented suspectcells. Images of the suspect cells and their relative position along theoptically transparent viewing surface of the chamber are recorded andarchived for later use. Since ICC imaging alone lacks the specificity toassess the clinical significance of blood samples with less than 5 CTC'sor to provide detailed genetic information about suspected cancer cells,subsequent analysis using multiparametric genetic profiling onindividual suspect cells is needed to provide a complete profile andestablish a confirmatory mechanism that can be used in diagnosticanalysis, including screening, assessing recurrence of disease, andoverall survival. One embodiment of the present invention utilizesfluorescent in sit hybridization (FISH) as a multiparametric geneticanalysis, but other profile assessments are considered. This providesboth phenotypic and genotypic profile assessment for an individual cellpresent along the viewing surface

FISH requires temperatures above the melting temperature of DNA as wella reagents that are not compatible with the ICC labeling. Most of theICC and DNA labels do not survive the FISH procedure with any signalslost in processing. Thus, a cell that was identified as being aninteresting cell for FISH analysis can not be traced back on itsposition. Therefore there is a need to have a detection method that oncethe ICC image is obtained, the cell position along the opticallytransparent viewing surface is maintained for subsequent multiparametricgenetic analysis (FISH) or other types of analysis in which the ICClabels are lost. This is achieved, in part, by fixing the cells on theoptically transparent surface after the ICC image is obtained without aloss of cells or any substantial movement along the surface. Accordinglyafter addition of the FISH reagents, the cartridge is placed on ahotplate having the surface with the immobilized cells in contact withthe hotplate. Depending on the type of assay the hotplate is programmedwith different temperature cycles that run between 2 and 48 hours. Afterthe temperature cycles are completed, the excess FISH reagents areremoved from the cartridge. The cartridge is filled with a buffersolution containing a DNA label to visualize the nuclei of immobilizedcells. Depending on the DNA label used, the label remains in thecartridge or is washed out of the cartridge after staining.

Next, the cartridge is placed back in the CellTracks® Analyzer II Systemfor a second scan. Because cells present on the upper surface during thefirst ICC image analysis were immobilized, the same cells are still inthe same relative location inside the cartridge. To assess the shift ofthe cartridge relative to the imaging system (CellTracks® Analyzer IISystem), the locations of the nuclei in the images of the second scanare compared to the location of the nuclei in the images of the firstICC scan. The shift of these images with respect to each other isdetermined using convolution algorithms. After this shift has beendetermined a specific cell of interest, based on its ICC image, can beselected from a list and be relocated on the surface of cartridge afterFISH in the second scan. Next fluorescent images of the different FISHprobes are acquired.

FIG. 9 shows a representative image of a tumor cell, identified by ICCand probed for the presence of chromosome 1, 7, 8 and 17. Panel A showsa list of CTC candidates identified by the software as cytokeratin(cytoskeletal protein present in cells of epithelial origin) positiveand DAPI (nucleic acid stain) positive. The corresponding images of thehighlighted event were identified as a CTC by the user as it confirmedthe CTC definition (cytokeratin positive, CD45 negative, DAPI positiveevent with the morphological appearance of a cell). Four images takenwith a 10× objective are shown in Panel B. The top left image shows theDAPI staining of the nucleus and the bottom left image the cytokeratinstaining of the cytoplasm. CD45 staining and FITC staining are lackingas illustrated by the lack of positive staining. After the cells werepreserved and probed for the centromeric probes for chromosome 1, 7, 8and 17, images of the upper surface of the cartridge were reacquired andthe fluorescent signals of the probes for chromosome 1, 7, 8 and 17 areshown in Panel C for the same cell shown in B. Two copies of chromosome1, three copies of chromosome 7, four copies of chromosome 8 and twocopies of chromosome 17 are clearly visible demonstrating that the cellis aneuploid and confirming that the cell indeed is a cancer cell.

Images displayed in FIG. 10 are acquired using a 10×, NA 0.5 planachromat objective. Although the resolution is sufficient for mostcentromeric probes, it is not sufficient for the gene specific probes,for example HER-2 and EOFR FISH probes. For this reason the 10×, NA 0.5objective, the objective used for ICC image acquisition, is replaced bya 40×, NA 0.6 objective, corrected for the optical thickness of thetransparent upper surface of the cartridge. The use of high NAobjectives allows for 3D imaging of the cell of interest allowing for aconfident determination of the correct number of copies for FISH probelabeled sequence in a selected cell. Multiple images at different focalplanes along the optical axis of a specific cell of interest areacquired followed by 3D reconstruction of the cell. FIG. 10 shows 5 suchslices through the cell using excitation/emission filters for 5different fluorochromes. In Panel A, five slices for PE are shown. Inslice #2, only two signals are visible whereas in slice #3 three signalsare visible. Panel B shows 5 slices of the DAPI staining. In the APCslices of Panel C, #2 slice shows 1 signal. In the FITC slices of PanelD, slice #3 shows two signals and slice #4 shows two different signals,making the total for this probe 4. In Panel E, Dy415 slices show 1signal in slice #2 and two signals in slice #3. From the images, it isclear that the probes are located in different parts of the nucleus andthat using only one focal plane the counting of the signals would not becorrect. In FIG. 11 Panel A fluorescence ICC images of the CTC,presented as a stack of images from FIG. 10. In panel B, the slices foreach fluorochrome are added and the average intensity is presentedthereby scaling the images to use the full range of intensity levelswhich facilitates enumeration. The count for the number of signals witheach probe are shown next to each image (i.e. 4 for PE, 1 for APC, 4 forFITC and 2 for dy415).

It is understood that the subject matter of this invention is notlimited to the detection of cancer cells but can also be used tocharacterize other cell types. One cell type frequently pursued fordetection of cytogenetic abnormalities is fetal cells in maternal blood.To enrich for such cells, markers need to be targeted that are presentat high frequency on the fetal cells and in low frequency on thematernal cells. One cell type that is frequently pursued is thenucleated red blood cells. A marker that is present on all nucleated redblood cells is, for example, the transferrin receptor (CD71). Whencoupled to ferrofluids, nucleated red blood cells are reproduciblyenriched from whole blood with the CellTracks® Autoprep® System. Theenriched cells contain fetal nucleated red blood cells, maternalnucleated red blood cells, activated T-lymphocytes, immaturereticulocytes and other cells that have been carried over by theimmunomagnetic enrichment. The enriched cell population is now bestained with markers that discriminate between cells of fetal andmaternal origin. One such panel of markers is the use of CD45 toeliminate leukocytes from the analysis in combination with Hemoglobin Fthat is present in fetal red blood cells but only rarely in maternal redblood cells, carbonic anhydrase that is only present in adult red bloodcells and DAPI to identify the nucleus of the cells. The CellTracks®Autoprep® System is used to stain the cells in a reproducible manner. Assome of the antigens are intracellular the cells, cells need to bepermeabilized for the antibodies to pass the cell membrane. The agentsused for permeabilization also lyse the immature reticulocytes,specifically selected by the use of CD71 and the remaining erythrocytesthat were carried over through the procedure. After the staining of thecells with the probes that have different reporter molecules, thecartridge containing the stained cells are placed in the CellTracks®Analyzer II System. The system identifies fetal nucleated red blood cellcandidates as DAPI⁺, CD45⁻, Fetal Hemoglobulin⁺, Carbonic Anhydrase⁻events. The user can confirm that these events indeed have all thecharacteristics typical for fetal nucleated red blood cells. After thesystem remembers the location of the fetal nucleated red blood cells,the cartridge is emptied as described above and the cells are hybridizedwith probes for cytogenetic analysis. Probes that are typically used toidentify relatively frequent cytogenetic abnormalities are those thatrecognize chromosome X, Y, 13, 18 and 21. After the cells have beenstained, the cartridge is reinserted in the CellTracks® Analyzer IISystem, because the cells that were present on the upper surface duringthe first ICC image analysis were immobilized the same cells are stillon the same location inside the cartridge. The system returns to theevents and takes images of the fluorochromes used to identifychromosomes X, Y, 13, 18 and 21. The user than assesses whether the copynumber of each of the chromosomes and determines the sex of the fetusand whether or not the copy number of the chromosomes suggest thepresence of cytogenetic abnormalities.

Example 1—Detection of Cytogenetic Aberrations after CTC Identification

CTCs from 7.5 mL of blood were identified as cytokeratin+, CD45−nucleated cells after immunomagnetic enrichment targeting the EpCAMantigen using the CellSearch System (Veridex, LLC). CTCs are identifiedby the CellTracks® Analyzer (Immunicon Corporation) where the cells aremagnetically held along the upper surface of a cartridge. Forcytogenetic analysis, the fluid in the cartridge was removed and thecells fixed while maintaining their original position. Fluorescentlylabeled probes for chromosome 1, 7, 8 and 17 were introduced into thecartridge and hybridized to the cells. The fixation and hybridizationprocess removes the fluorescent labels used for CTC identification.After hybridization the cartridges were again placed on the CellTracks®Analyzer and analyzed for a second time. The fluorescent images of theCTC's identified in the first scan are then combined with thefluorescent images from each of the four chromosomes labels obtained inthe second scan. The number of chromosomes 1, 7, 8 and 17 wereenumerated for each CTC that was identified in the first scan. Thenumber of chromosomes detected in leukocytes that surrounded the CTC'swere used as internal controls. In 7.5 mL of blood from 8 patients withmetastatic carcinoma, 1 to 7 CTC's were identified. Greater than or lessthan two copies of chromosome 1, 7, 8 or 17 were detected in all 8patients. Heterogeneity in the chromosomal abnormalities were not onlydetected between CTC's of different patients but also among CTCs of thesame patient. Of the 21 CTCs examined, 77% showed chromosomalabnormalities and a majority showed an increase in the number of copiesof the chromosomes. In contrast, more than 80% of the leukocytesexamined showed two copies of the chromosomes and none showed anincrease in chromosome copy number.

Conclusions: Cytogenetic composition of CTC's can be assessed after theyhave been identified. The presence of aneusomic CTC's providesinformation to the outcome of patient conditions and provides aprognostic indicator of clinical outcome. Further, gene alterations inCTC's provide indices to current and future cancer therapies.

Example 2—Evaluation of Anti-Cancer Targets on CTC's to PredictTherapeutic Success

The CellSearch System™ has been used in multi-center prospective studiesto demonstrate that presence of tumor cells in blood of patients withmetastatic carcinomas is associated with poor survival prospects.Failure to eliminate Circulating Tumor Cells (CTCs) after one cycle oftherapy in these studies strongly suggests that these patients are on afutile therapy. Assessment of the presence of therapeutic targets on thetumor should enable the appropriate choice of therapy. Anti-cancertargets are identified on CTCs before initiation of therapy. Cells from7.5 mL of blood are identified as cytokeratin(CK)+, CD45− and nucleatedafter EpCAM immunomagnetic selection. Suspect CTCs are identified andlocalized at the upper surface of a cartridge where they are held by amagnetic field. Fluorescently labeled antibodies that recognizetreatment targets associated with known therapies such as HER2, IGF-1,Bcl-2 and EGFR are assessed on the CTCs. Subsequently, CTCs arepreserved for cytogenetic analysis. After the fluid in the cartridge isremoved, the cells are fixed and maintain their original position forprobe hybridization. Since the system knows their original position, thecells can be reexamined for the presence of probes of interest. Theresults show a CTC and a leukocyte before and after hybridization withchromosome 1, 7, 8 and 17.

We claim:
 1. A device for preparing a population of suspect cancer cellsfor fluorescent in situ hybridization comprising: a. a syringe pump fortransfer of fluids in cartridge, wherein said fluids are buffer,fixative, or hybridization buffer; b. a sample engagement system forreagent transfer; c. an aspiration probe for drying; and d. a heaterblock wherein said block operates through a feedback thermocouple,control thermocouple, and temperature controller.